1. Field of the Invention
This invention relates generally to gene expression in normal and neoplastic cells, and specifically to a novel tumor suppressor gene, DPC4.
2. Description of Related Art
The development of human cancer involves the clonal evolution of cell populations which gain competitive advantages over other cells through the alteration of at least two distinct classes of genes: proto-oncogenes and tumor suppressor genes (Weinberg, R. A., Science, 254:1138, 1991; Bishop, J. M., Science, 235:305, 1987). Tumor suppressor genes are characterized by alterations which inactivate both original alleles (Knudson, J. A. G., Cancer Res., 45:1437, 1985). Many tumor suppressor genes are inactivated by intragenic mutations in one allele accompanied by the loss of a relatively large chromosomal region containing the other gene copy. This loss of one copy of a region or a gene is also called allelic loss or loss of heterozygosity (LOH). The finding of a homozygous deletion within a region suffering a high frequency of LOH, has been crucial to the discovery of several tumor suppressor genes (Dryja, et al., Proc. Natl. Acad. Sci. USA, 83:7391, 1986; Fearon, E. R., et al., Science, 247:49, 1990; Kamb, et al., Science, 264:436, 1994) due to the relatively small sizes of the homozygous deletions.
Genetically, the absence of a gene or its function can occur through biallelic inactivation (Knudson, et al., Cancer Res., 45:1437, 1985). One mode of biallelic inactivation involves the combination of an intragenic mutation of one allele together with the loss of a relatively large chromosomal region containing the second allele (LOH). A second mechanism for biallelic inactivation is a combination of two inactivating point mutations targeting a specific gene. A third involves homozygous deletions. Homozygous deletions are thought to be the result of two steps, the loss of a larger chromosomal region and the separate loss of a considerably smaller area.
Knowledge regarding the frequency of homozygous deletions is very limited. They are generally small regions of a chromosome, and probably most deletions are smaller than 2 Mb in size. Two techniques are available for direct searches of homozygous deletions. One technique is the mapping of a preselected chromosomal area with markers. The dramatic increase of the number of sequence tagged sites(STSs) assigned to individual human chromosomes in recent years makes this approach increasingly productive. Examples for the successful application of this technique are the discovery of genes such as RB1, DCC and MTS1 (Kamb, et al., supra; Dryja, et al., supra; Fearon, E. R., et al., supra). A more elegant way to detect homozygous deletions, without the prior limitation of a preselected chromosomal region is the technique of representational difference analysis (RDA) (Lisitsyn, et al., Science, 259:946, 1993). A number of candidate loci have been identified to date by RDA, including one within the region of the BRCA2 gene (Schutte, et al., Proc. Natl. Acad. Sci., USA, 92:5950, 1995; Lisitsyn, et al., Proc. Natl. Acad. Sci., USA, 92:151, 1995).
The molecular events known in pancreatic ductal tumorigenesis include the acquisition of K-ras mutations in over 80% (Almoguerra, et al., Cell 53:549, 1988; Hruban, et al., Am. J Pathol, 143:545, 1993; Redston, et al., Cancer Res., 54:3025, 1994), mutations of the p53 gene in 50-70% (Redston, et al., supra; Pellegata, et al., Cancer Res., 54:1556, 1994), and mutations or homozygous deletions of the p16 gene in over 85% (Caldas, et al., Nat. Genet., 8:27, 1994). Allelotype data suggested the existence of additional tumor suppressor genes at other loci (Hahn, et al., Cancer Res., 55:4670, 1995; Seymour, et al., Cancer Res., 54:2761, 1994). One such interesting location is chromosome 18q, lost in nearly 90% of pancreatic cancers (Hahn, et al., supra; Seymour, et al., supra; Griffin, et al., Cancer Res., 55:2394, 1995). A candidate suppressor gene, DCC, is included in the involved region of 18q LOH, but due to the length and complexity of the gene (29 exons spanning 1.4 megabases), sequence analyses have not been performed in pancreatic carcinomas nor many other tumor types (Cho, et al., Genomics, 19:525, 1994).
Chronic ulcerative colitis (UC) and Crohn disease (CD) are associated with an increase risk of colorectal neoplasia. In most studies, the spectrum and frequencies of genetic alterations in these neoplasms have not been distinctly different from those seen in sporadic colorectal adenomas and carcinomas. Thus, K-ras mutations, 17p deletions and p53 mutations, 18q deletions and APC mutations have been identified at significant rates (Kern, et al., Gastroenterol, 107:420, 1994; Redston, et al., Gastroenterol., 108:383, 1995; Burmer, et al., Gastroenterol., 99:416, 1990; Meltzer, et al., Cancer Res., 50:3627, 1990; Bell, et al., Br J Cancer, 64:174, 1991; Chen, et al., Gastroenterol., 102:1983, 1992; Burmer, et al., Gastroenterol., 103:1602, 1992; Yin, et al., Gastroenterol., 104:1633, 1993). One candidate gene on 18q is the DCC gene, but DCC, as discussed above, is among the largest genes known and mutational analyses have been difficult (Fearon, et al., supra). Thus the target gene for 18q deletions in colitis-associated neoplasia has remained unknown.
Currently, there is one example of a potential therapeutic target which is localized to a hotspot of homozygous deletions at the p16 gene locus. This deletion at 9p21, found in various tumor types, often includes the MTAP (methylthioadenosine phosphorylase) gene (Olopade, et al., Proc. Natl. Acad. Sci., USA, 92:6489, 1995). The 9p21 region is homozygously deleted in 40% of pancreatic cancers (Caldas, et al., Nature Genet., 8:27, 1994), and half of these deletions include the MTAP gene. This purine salvage pathway member is therefore part of the biochemical difference of certain tumor types and represents a target for a chemotherapeutic intervention. This is an example of how the selective absence of a functional copy of a gene contained within a homozygous deletion in cancer might provide a more specific approach to chemotherapy by potentially facilitating the development of drugs toxic to the tumor cell while remaining non-toxic to normal cells. In the case of pancreatic cancer, the frequent homozygous deletions of 9p21 and 18q provide large regions in which to search for potential tumor-specific therapeutic targets.